In the next funding period of this grant we will continue our studies on the structural constraints of T cell recognition of peptide and non- peptide antigens in the context of class I and class II MHC and extend them to a disease model. Our first specific aim is a direct continuation of studies initiated in the last funding period on a class II restricted, CD4+ T cell response of high precursor frequency in naive mice to a structurally rigid molecule, heme. In order to complete our studies on this unusual response we wish to determine the mechanism by which heme generates an I-A restricted polyclonal response. Our favored hypothesis is that by changing the set of peptides bound to syngeneic MHC, heme creates novel antigenic structures that, by analogy to allogeneic responses, stimulate a wide variety of T cells in the peripheral repertoire. We will thus determine whether the composition of the population of naturally processed peptides that are normally found associated with I-A molecules is changed in the presence of heme and whether heme does this by directly binding to MHC. In addition, we will test an alternate hypothesis, that heme-reactive T cells have been previously expanded in vivo. Our second specific aim is to examine the conformational constraints of peptide/MHC interactions using a novel approach which we have developed for examining antibody/antigen interactions i.e. H-D exchange and 2D NMR. Initially we will use K/b and the VSV-8 and SEV-9 peptides, for which there are known crystal structures, to facilitate data interpretation. We will then examine one of the peptide/MHC interactions we have extensively characterized in the last funding period of this grant; the cyt c peptide 41-49 which has the unique ability to bind to both K/b and D/b molecules. Our third specific aim applies molecular approaches to investigating the mechanism of immune regulation of the auto-immune disease experimental allergic encephalomyelitis (EAE). We will determine whether naturally processed TCR peptides are found associated with the EAE high-responder H- 2/u class I MHC molecule on the surface of an anti-MBP (myelin basic protein) T cell hybridoma. Identified peptides will be tested for their ability to induce CD8+, class I restricted anti-TCR regulatory cells using an in vitro peptide priming system.